Aspergillus fumigatus is pathogenic fungus that humans inhale on a daily basis. The ability of the respiratory innate immune system to prevent the germination of inhaled conidia (vegetative spores) into tissue-invasive hyphae represents a critical immunologic checkpoint. Due to advances in medical technology, there has been a significant rise in patients that live in immune compromised conditions, leading to an estimated 200,000 cases of aspergillosis annually. In high-risk groups, mortality rates remain 25-50% despite contemporary combination antifungal therapies. These trends highlight the need for an improved understanding of the molecular and cellular basis of sterilizing immunity to advance immune-based adjunctive approaches. In the initial funding period of this project we introduced a novel fungal bioreporter, termed fluorescent Aspergillus reporter (FLARE), to trace the viability of conidia during cellular interactions with immune cells in the lung. Using this approach, we identified spleen tyrosine kinase (Syk) as essential for innate defense in the lung, and in pilot studies, show that Syk acts in a cell-extrinsic manner to regulate neutrophil fungicidal activity. Although Syk can integrate signals from C-type lectin receptors, Fc receptors, and integrins in myeloid cells, its essential cellular source and fungus-induced activation signals during respiratory fungal challenge remain poorly defined. Beyond neutrophils, our studies show that -CC- chemokine receptor 2 (CCR2)-expressing inflammatory monocytes (IMs) form a second essential innate effector cell during respiratory challenge. In the lung, IMs differentiate into monocyte-derived dendritic cells (Mo-DCs), coincident with direct conidial uptake and killing, and engage in intercellular crosstalk with lung neutrophils to boost antifungal activity in the lun. In pilot studies, transcriptional profiling of IM/Mo-DCs and lung proteomic analyses identified fiv candidate mediators of intercellular crosstalk and the respiratory burst as a candidate effector mechanism. During the next project period, we propose to gain a deeper understanding of IM/MoDC - neutrophil intercellular crosstalk, its induction and regulation by Syk activity, its relevant molecular transmitters, and its role in anticonidial defense in the lung. In Aim 1, we identify protective mechanisms of Syk signaling by identifying its essential cellular source and by determining essential upstream adaptor proteins and receptors that mediate conidial uptake and killing in the lung. In Aim 2, we will define reciprocal regulation of IM/Mo-DC and neutrophil antifungal activity by establishing the cellular and trafficking requirements, induction signals, ad directional nature of intercellular crosstalk. In Aim 3, we will define molecular transmitters and effector systems of IM/Mo-DC-neutrophil crosstalk. The proposed studies are significant and innovative because they identify innate immune crosstalk between IMs and neutrophils as a novel mechanism to regulate antifungal immunity in the lung. Understanding the induction, regulation, and participants of innate immune crosstalk addresses a critical knowledge gap that will inform immune-enhancing strategies in vulnerable patient groups.